Biomedical imaging plays a central role in a large number of diagnostic and therapeutic procedures including visualizing external and internal anatomical and physiological structures, features, and systems; evaluating complex biological events in the body at the organ, tissue, cellular, and molecular levels, and facilitating image guided surgery techniques. Imaging allows physicians and other health care professionals to detect and diagnose the onset of disease, injury, and other disorders at an early stage and to accurately monitor progression or remission of a condition. Biomedical imaging also enables delivery of targeted and minimally invasive therapies for treating and managing a range of conditions. A large number of applications of biomedical imaging have matured into robust, widely used clinical techniques including planar projection and tomographic x-ray imaging, magnetic resonance imaging, ultrasound imaging, and gamma ray imaging.
Biomedical images are generated by detecting electromagnetic radiation, nuclear radiation, acoustic waves, electrical fields, and/or magnetic fields transmitted, emitted and/or scattered by materials, where the materials can be biological materials and/or materials introduced in the body such as implants, contrast agents, infusions, tracers, etc. Modulation of energy (e.g., radiative, acoustic, etc.) and/or particles provided to a sample via interaction with materials such as biological molecules and tissue structures yields patterns of transmitted, scattered or emitted radiation acoustic waves, electrical fields or magnetic fields that contain useful anatomical, physiological, and/or biochemical information. Modulation may occur via mechanisms involving interactions of endogenous materials in the sample and/or mechanisms involving interactions of exogenous imaging agents introduced to a sample to enhance the usefulness of the acquired image, such as contrast agents, dyes, optically or radiolabel materials, biomarkers, and other agents. Biomedical imaging has been demonstrated as generally useful for providing images of surface and subsurface components of tissue samples and also provides a means of real time monitoring of components of biological samples, in vivo and in vitro.
Advanced optical imaging methods, such as confocal scanning laser tomography and optical coherence tomography, have emerged as valuable molecular imaging techniques for visualizing biological processes at a cellular and subcellular (e.g., molecular) levels. Established optical molecular imaging techniques are based on monitoring spatial variations in a variety of optical parameters including the intensities, polarization states, and frequencies of transmitted, reflected, and emitted electromagnetic radiation. Given that many biological materials of interest are highly turbid with respect to most frequencies in the ultraviolet and visible regions of the electromagnetic spectrum, research is currently directed to developing and enhancing imaging techniques using near infrared excitation radiation from about 700 nanometers to about 1200 nanometers corresponding to an “optical window” present in many of these materials. Electromagnetic radiation of this wavelength range is capable of substantial penetration (e.g., up to a millimeter) in many biological materials and is considerably less phototoxic than visible and ultraviolet electromagnetic radiation. Infrared optical molecular imaging techniques, therefore, offer the promise of providing nondestructive and noninvasive imaging of subsurface biological structures in biological samples.
Recent advances in high intensity, mode locked near infrared laser optical sources make nonlinear optical imaging methods, such as multiphoton (MP) microscopy and second harmonic generation (SHG), an important class of infrared molecular imaging methods for visualizing cellular and subcellular structures in biological samples. Nonlinear imaging techniques are particularly useful for providing high resolution images for probing physiology, morphology, cellular microenvironments, and cell-extracellular matrix and cell-cell interactions in intact tissues and living organisms. MP microscopy uses a high intensity, temporally short laser pulse to provide highly localized nonlinear excitation of fluorescence. In two photon fluorescence excitation techniques, for example, absorption of two lower energy photons simultaneously excites an electronic transition in a fluorophor, thereby causing radiative decay resulting in fluorescence emission of a single higher energy photon. As the probability of two photon absorption is relatively low (for example, as compared to single photon absorption), excitation in this technique is limited to a spatially confined focused region of the excitation beam having a sufficiently high intensity of photons. Second harmonic generation, in contrast, does not arise from an absorptive process. Rather, the second harmonic phenomenon results from a nonlinear scattering interaction of radiation with a non-centrosymmetric environment of a sample. In this technique, an intense laser field is provided to the sample that induces a nonlinear, second order, polarization in the spatial orientation of molecules exposed to the excitation radiation. The induced polarization results in generation of a coherent wave having a frequency that is exactly two times that of the incident excitation radiation. In both MP microscopy and SHG, a two dimensional image is typically generated by detecting fluorescence or polarized light, respectively, while the excitation beam is systematically scanned across a given layer of the sample. Three dimensional images are formed by scanning a plurality of layers at different depths.
A number of advantages are provided by nonlinear techniques relative to conventional linear optical imaging techniques. First, these techniques are ideally suited for use of infrared excitation radiation, particularly having wavelengths in the optical window region from about 700 nanometers to about 1200 nanometers of many biological samples. Thus, nonlinear optical techniques are capable of penetrating and imaging many types of tissues and typically do not lead to significant photoinduced sample degradation during analysis. Second, nonlinear optical imaging methods are capable of providing images with enhanced axial resolution relative to conventional optical imaging techniques due to the highly localized excitation arising from the nonlinear dependence of excitation rate on illumination intensity. Third, some applications of nonlinear advanced optical techniques to biomedical imaging, such as second harmonic generation methods, do not require exogenous labeling/staining. These techniques, therefore, can eliminate the need for complex and invasive labeling procedures common to conventional optical molecular imaging methods. Finally, different nonlinear techniques may be combined and used in tandem to provide complementary information relating to tissue structure and composition. For example, the combination of MP and SHG images provides enhanced cellular and subcellular information, as each technique employs fundamentally different excitation processes and, thus, provides substantially different contrast mechanisms.
Given the demonstrated capabilities of nonlinear optical imaging techniques for probing cellular and subcellular morphology and composition, researchers are currently pursuing applications of these techniques for detecting, diagnosing, and monitoring the onset and progression of disease. Proposed applications of nonlinear optical imaging include diagnosis of cancer and in situ evaluation of angiogenesis and metastasis processes, and monitoring the progression of neurodegenerative diseases such as Alzheimer's disease. Although the potential for such applications, including endoscopy and optical biopsy applications, is clear, these techniques have not yet matured to the point so as to provide a robust clinical tool. To develop this, and other important applications of nonlinear optical imaging, histopathological features and structural motifs in biomedical images that correlate with specific disease conditions in human and animal patients must be identified and characterized, particularly as a function of the progression or remission of a disease. Further, enhancements are also need to transform the instrumentation used in nonlinear imaging techniques into a reliable instrument capable of implementation in range of clinical applications.